In recent years, the mechanism by which mammalian immune systems, such as human and murine systems react to infections, foreign antigens, and to so-called "self antigens" in connection with autoimmune diseases has begun to be established. See, in this regard, Grey et al., Scientific American 261(5): 56-64 (1989); Male et al., Advanced Immunology (J. P. Lippincott Company, 1987), especially chapters 6 through 10.
Well known, both to the skilled artisan and to the general public is the role of antibodies, sometimes referred to as "immunoglobulin" or the less correct and older "gammaglobulin" in response to infection. Antibodies are protein molecules which are produced by B cells in response to infection. It is well known that these antibodies act to "disable" or to inactivate infectious agents in the course of combating the infection.
In order for antibodies to be produced, however, preceding events must occur which lead to stimulation of the B cells which produce the antibodies. One of the key events involved in the processes leading to antibody production is that of antigen recognition. This aspect of the immune response requires the participation of so-called "T-cells", and is less well known than the antibody response commented on supra.
Briefly, and in outline form, antigen recognition requires interaction of an "antigen presentation cell", a "processed antigen", and a T-cell. See Grey and Male, supra. The "processed antigen", in an infection, is a molecule characteristic of the pathogen which has been treated, i.e., "processed", by other cells which are a part of the immune system. The processed antigen interacts with a receptor on the surface of an antigen presentation cell in a manner not unlike a lock fitting into a key hole or, perhaps more aptly, two pieces of a jigsaw puzzle.
The configuration of the complex of processed antigen and receptor on antigen presentation cell allows the participation of T-cells. T-cells do not join the complex unless and until the processed antigen has fit into the receptor on the antigen presentation cell. This receptor will hereafter be referred to by its scientific name, the major histocompatibility complex (MHC), or the human leukocyte antigen (HLA). Generally, MHC is used to refer to murine systems, and HLA to humans.
These receptors fall into two classes. MHC-II molecules are involved in most responses to pathogens. In contrast, MHC-I molecules are involved when the pathogen is a virus, or when a malignant cell is involved. When MHC-I participation is involved, there is no antibody stimulation; rather, the interaction of MHC-I, processed antigen and T-cell leads to lysis of cells infected with the pathogen.
The foregoing discussion has focused on the events involved in responding to "infection", i.e., the presence of pathogenic foreign material in the organism. Similar mechanisms are involved in autoimmune diseases as well. In these conditions, the organism treats its own molecules as foreign, or as "self-antigens". The same type of complexing occurs as described supra, with an antibody response being mounted against the organism itself. Among the diseases in which this is a factor are rheumatoid arthritis, diabetes, systemic lupus erythromatosus, and others.
The ability of the T-cell to complex with the processed antigen and MHC/HLA complex is dependent on what is referred to as the T-cell antigen receptor, referred to as "TCR" hereafter. The TCR is recognized as a heterodimer, made up of alpha (.alpha.) and beta (.beta.) chains. Five variable elements, coded for by germline DNA and known as "V.alpha., J.alpha., V.beta., D.beta., and J.beta." as well as non-germline encoded amino acids contribute to the TCR. See, in this regard, Marrack, et al., Immunol. Today 9: 308-315 (1988); Toyonaga et al., Ann. Rev. Immunol. 5: 585-620 (1987); Davis, Ann. Rev. Immunol. 4: 529-591 (1985); Hendrick et al., Cell 30:1 41-152 (1982). With respect to the binding of TCR with processed antigen and MHC, see Babbitt et al., Nature 317: 359-361 (1985); Buus et al., Science 235: 1353-1358 (1987); Townsend et al., Cell 44: 959-968 (1986); Bjorkman et al., Nature 329: 506-512 (1987).
Generally, both the alpha and beta subunits are involved in recognition of the ligand formed by processed antigen and MHC/HLA molecule. This is not always the case, however, and it has been found that so-called "superantigens" stimulate T-cells with a particular V.beta. element, regardless of any other element. See Kappler et al., Cell 49: 273-280 (1987); Kappler et al., Cell 49: 263-271 (1987); MacDonald et al., Nature 332: 40-45 (1988); Pullen et al., Nature 335: 796-801 (1988); Kappler et al., Nature 332: 35-40 (1988); Abe et al., J. Immunol 140: 4132-4138 (1988); White et al., Cell 56: 27-35 (1989); Janeway et al., Immunol. Rev. 107: 61-88 (1989); Berkoff et al., J. Immunol. 139: 3189-3194 (1988), and Kappler et al., Science 244: 811-813 (1989).
The "superantigens" mentioned supra, while generally stimulating T-cells, are somewhat specific in terms of the particular form of the V.beta. moiety which is present on the stimulated T cell. This feature is one aspect of the invention, i.e., the ability to assay for particular subtypes or subclasses of T-cells, based upon the cell surface antigens presented by these subclasses.
It must be pointed out, however, that the expansion of the V.beta. element does not in and of itself mean that a superantigen is involved, and in the discussion which follows, the inventors do not limit themselves to the possibility that vasculitic disorders are caused by a superantigen mechanism.
Staphylococcus antigens discussed supra are not the only substances which provoke a restricted, V.beta. proliferative response. Indeed, it is the purpose of this invention to show that such a response occurs, and can be measured in other diseases and conditions, vasculitic disorders in particular, even where an etiological agent is not recognized. It is the purpose of this invention to provide a method for diagnosing a vasculitic disorder by assaying a T-cell containing sample from a patient for a particular V.beta. element, and comparing the value determined to "base line" or "normal" values. Deviation from normal is indicative of the particular vasculitic disease associated with variations in the level of the particular V.beta. element. In a particularly preferred embodiment, the method is used for diagnosis of Kawasaki disease, but other vasculitic disorders, such as Wegener's Granulomatosis, Systemic Lupus Erythematosis, Behcet's disease, Chung-Strauss Syndrome, Lymphomatoid Granulomatosis, juvenile rheumatoid arthritis, Polyarteritis Nodosa, Rheumatic Fever, and Thromboangiitis Obliterans may also be diagnosed in the manner described herein.
In a particularly preferred embodiment, the V.beta. element is assayed for by utilizing polymerase chain reaction ("PCR"), as described in, e.g., U.S. Pat. Nos. 4,683,195; 4,800,159 and 4,683,202 the disclosures of which are incorporated by reference. Another preferred method involves assaying the V.beta. element by use of antibodies, monoclonal antibodies in particular.
The data which follow deal, for the most part with Kawasaki Disease, and to that end some discussion is necessary.
Sometimes referred to as monocutaneous lymph note syndrome, Kawasaki disease ("KD" hereafter) is an acute multi system vasculitis of unknown etiology. The disease primarily affects infants and young children. See Kawasaki, Jpn. J. Allergol 16: 178-222 (1967); Rauch et al., Pediatr. Infect. Dis. 4: 702-703 (1985). While KD does occur worldwide, it is most prevalent in Japan and in children of Japanese ancestry. Primary clinical manifestations include prolonged fever, bilateral non-exudative conjuctivitis, induration and erythema of extremities, inflammation of lips and oropharynx, polymorphous skin rash, and cervical lymphadenopathy.
In Japan and in the United States, KD has become one of the most common causes of acquired heart disease in children. Recent studies have shown that when gamma globulin is administered intravenously ("IVGG") during the acute phase of the disease, coronary artery lesions, which otherwise develop in 15-25% of patients, are significantly decreased. See Newburger et al., N. Engl. J. Med. 315: 341-6 (1986); Nagashima et al., J. Pediatr. 110: 710-2 (1987); Firisho et al., Lancet ii: 1055-57 (1984); Rowley et al., J. Pediatr. 113: 290-94 (1988); Newburger et al., N. Eng. J. Med. 324: 1633 39 (1991). Thus, in order to treat this disease effectively, as with all other vasculitic diseases, early recognition is essential.
KD is characterized by an acute stage, as well as a convalescent stage. These are described in some detail infra. The acute phase is characterized, inter alia, by marked immune activation. Investigators have demonstrated, for example, increased numbers of circulating and infiltrating T cells bearing the HLA-DR activation antigen and elevated serum soluble IL-2 receptor levels. The phenomena are indicative of T-cell activation. See Leung et al., J. Clin. Invest. 79: 468-472 (1987); Terai et al., Hum. Pathol. 21: 231-234 (1990); Lang et al., J. Pediatr. 116: 592-596 (1990). In addition, acute KD has been associated with increased production of IL-1.beta., TNF-.alpha., IL-6, IL-2, and IFN-.gamma.. See, e.g., Matsubara et al., Clin. Immunol. Immunopathol 56: 29-36 (1990); Maury et al., J. Lab. Clin. Med. 113: 651-54 (1989); Lang et al., J. pediatr. 115: 939-43 (1989); Leung et al., Lancet ii: 1928-1302 (1989); Rowley et al., Ped. Inf. Dis. J. 7: 663-67 (1988); Ueno et al., Clin. Exp. Immunol 76: 337-342 (I989); Jordan et al., in Kawasaki, ed., The Third International Kawasaki Disease Symposium 1989: 144-46. The cytokines referred to supra are believed to play a significant role in the pathogenesis of vascular cell injury during acute KD, due to their proinflammatory and prothombotic effect on endothelial cells. See Mantovani et al., Immunol. Today 10: 370-74 (1989). Vascular endothelia, in KD lesions, have been demonstrated to express cytokine inducible leukocyte adhesion molecules known to be involved in localization of inflammatory cells. See Leung, supra. Patients with acute KD have been found to have cytotoxic antibodies against IL-1.beta., TNF-.alpha. and IFN-.gamma. stimulated endothelial cells, but not unstimulated cells. See Leung et al., J. Clin. Invest. 77: 1428-35 (1986); Leung et al., J. Exp. Med. 164: 1958-72 (1986).
As indicated, the cause of acute KD and the large scale immune response are unknown. While epidemiologic studies directed toward identifying potential environmental toxins, and laboratory culturing of body fluids for known microbial agents have taken place, an etiological agent has not been found. See Rauch et al., Ped. Infect. Dis. J. 6: 1016-21 (1987). Due to the acute, self-limited nature of the disease, geographic clustering of outbreaks, clinical symptoms of fever and eruptions which mimic conditions and diseases such as measles, roseola, and scarlet fever, as well as the unique susceptibility of young children, it has been suggested that humoral immunity develops early in life. KD is rarely seen over the age of 8, suggesting that there is an asymptomatic infection caused by a ubiquitous agent, followed by development of protective immunity in the general population.
The general observations on KD, as well as other vasculitic conditions, suggest that these diseases may have some similarities with disorders characterized by response to a so-called "superantigen". The patent applications cited supra and incorporated by reference herein, show that various superantigens lead to expanded populations of V.beta. elements or TCRs ("T cell receptor molecules"). This evidence is also presented in, e.g., Choi et al., J. Exp. Med. 172: 981-84 (1990); Kappler et al., Science 24: 811-13 (1989); and Choi et al, Proc. Natl. Acad. Sci. 86: 8941-45 (1989). The disclosures of these three references are all incorporated by reference herein. The superantigens, including bacterial toxins, provoke marked activation of T cells and monocytes/macrophages. For example, staphylococcal enterotoxins and streptococcal erythrogenic toxins induce IL-1 and TNF-.alpha. from monocytes. Staphyloccal enterotoxin and SPE-mediated stimulation of monocytes is a consequence of binding and transducing a positive signal through MHC-II molecules on monocyte cell surfaces. In the presence of antigen presenting cells, superantigens stimulate T-cell proliferation by selective stimulation of T cells expressing particular V.beta. elements. For example, Staphyloccocal TSST-1 stimulates T cells presenting V.beta.2. Choi et al., J. Exp. Med. 172: 981-4 (1990), have shown expansion of V.beta.2 cells in toxic shock syndrome. The similarities thus suggest at this time that vasculitic diseases especially KD, may involve the same phenomena as is involved in superantigen caused diseases and conditions, but, as noted supra, this is a theory rather than a known mechanism, as compared to the expansion of the V.beta. subtype, which is an empirical phenomenon. As noted supra, the inventors do not imply that vasculitic disorders are caused by superantigens.